Effect of zinc-deficient diet on oral tissues and periodontal indices in rats.

Zinc (Zn) as a nutritional factor affects the health of the oral tissues. This study was done for the evaluation of the effects of zinc deficiency on the oral tissues of rats. The study was carried out on 14 male Wistar rats, cessation of lactation on the 24(th) day after birth. The rats were randomly divided into two groups. Zinc deficient (ZD) diet was used for one group and another group was fed with a zinc-containing (ZC) diet. The alterations of the oral tissues in both groups were evaluated clinically after four weeks. Also the gingival index and periodontal pocket depth were recorded. The measurement of serum zinc level was done by atomic absorption spectrophotometry. The microscopic slides of oral tissue specimen were evaluated quantitatively. The serum zinc level of the ZD rats was lower than the ZC group (p< 0.001). According clinical findings, the gingival index was lower in ZC rat (p=0.001), but there was no significant difference regarding the periodontal pocket depth between two groups (p=0.07). Aphthous ulcer was observed in ZD rats on the floor of the mouth. There was no significant difference regarding the epithelial and keratin thickening between two groups. This study indicated that oral and periodontal health was better in ZC rats than in ZD rats. Aphthous lesions were more prominent in ZD rats. This study confirmed that zinc deficiency may endanger oral and periodo ntal structures.

serum level of Zn under 83 µg/dl is considered as Zn deficiency (6).

The current Dietary Reference Intakes (RDAs)
for zinc has recommended 11 mg for an adult male and 8 mg for an adult female daily (7).
A prominent feature of Zn deficiency is the broad range of produced pathologies and a spectrum of clinical manifestations, including impaired growth, alopecia, anemia, dwarfism, impaired sexual development, dermatitis, loss of hair, poor appetite, abnormal dark adaptation, delayed wound healing and mental lethargy (3,4,(8)(9)(10)(11)(12)(13). Zn is supposed to be utilized in the management and chemoprevention of cancer (14).
Many studies in limited number of cases and animals have been published to determine the effect of topical use of mouthrinse or toothpaste with Zn plus other anti-plaque and anti-gingivitis agents on teeth plaque (15)(16)(17)(18).
In 2007, Orbak reported, for the first time, that yhyperkeratinization was more prominent in Zndeficient rats. They suggested that Zn deficiency is a potential risk factor for oral and periodontal diseases (19).
In 2009, Üçkardeş reported that oral Zn supplementation improved the plaque Index and contributed to the prevention of dental caries in primary school healthy children with low socioeconomic level (17). The general manifestations of Zn deficiency have been reported in a number of studies (4,12,(20)(21)(22), but the effect of Zn deficiency on oral tissue has been described only in a few of studies (8,19,23). In this study, we investigated the changes in the oral tissues of Zndeficient rats.

Study setting
This investigation was carried out at the Wistar rats with cessation of lactation on the 24 th day of birth were used. The rats were randomly divided into two equal groups: One group was fed with a Zn-deficient diet (ZD), and another group

Statistical analysis
Statistical evaluation was performed using the Student's t-test for comparing the two groups.

Results
This investigation was carried out on 14 male rats divided into two groups with seven rats in each group. One Group was fed with a ZD diet, and the other group was fed with a ZC diet. The first observation of appetite reduction, loss of hair, diarrhea, and ulcerations of the skin and mucosa, in ZD rats occurred on the fifth day of the study and continued until the end of the experiment.

Body weight
The rats' weight was approximately equal at the beginning of the investigation and there was no statistical difference between them (P=0.62). At the end of the study, body weight was reduced in ZD rats, but was advanced in rats fed with a ZC diet with no significant difference (P=0.09) (Table1).

Serum level of Zn
The serum Zn level of the ZD rats was lower than the controls (ZC rats) (P< 0.001) ( Table 1).

Oral Manifestations
The number and surface area of aphthous ulcer is shown in Table 2. In the present study, aphthous ulcer was observed on the floor of the mouth with a high rate of 33.3% and the greatest average surface area of the ulcers was observed on the ventral side of the tongue (2.20±0.86). Although aphthous ulcer was often seen in the ZD group, it was not found in the ZC group.

Periodontal features
The mean gingival index for control group was lower than the ZD group (P = 0.001). Although the mean pocket depth in ZD group was higher than the control group, no significant difference, as regards to pocket depth, was found between the two groups of rats (P=0.07) (Tables 1).

Histopathological findings
Histological findings were as follows ( Figures   1 and 2). Epithelial thickening on the dorsal and  ventral surface of the tongue and on the palate in ZD rats was more than the ZC group and on the buccal mucosa and floor of the mouth in ZC group was more than the ZD group. There was no significant difference regarding epithelial thickening ( Figure 3). The mean keratin thickness on the dorsal surface of the tongue, buccal mucosa and palate in Zn-deficient rats was more than the ZC group and on the ventral surface of the tongue and floor of the mouth in ZC group was more than the ZD group. There was no significant difference regarding keratin thickness between groups ( Figure   4). Hyperkeratinization was much more prominent between the papillae on the dorsal surface of the tongue.

Discussion
Zn is an important mineral, essential for plants and for normal growth of rats and mice (1). Studies with rats therefore provide a useful model for investigating the effects of dietary Zn deficiency on oral tissues (19).   Only a few studies presented oral manifestations in rats with ZD diet (10,19). In addition, it has been reported that Zn deficiency might produce marked effects on about all components of the immune system (22,24 (19).